CHAPTER -I

INTRODUCTION

Since the mid-19th century, pharmaceuticals have moved from the periphery to

the center of health care. In the course of that transition, a new industry sector

expanded to global scope, the field of medicinal chemistry rose to its current

prominence, and governments adopted dual roles of supporting basic research and

regulating drug safety and efficacy. We are all patients at various points in life and for

patients drugs have taken on new medical roles and value. In recent years especially, it

has become common for people to take drugs for years, even decades, to reduce risks

of disease and increase life span. Taking drugs for life has intensified a long-standing

hybrid of scientific, emotional, and policy issues around side effects and widespread

resentment of companies that profit from drug invention and marketing It is therefore

of interest to have methodologies that allow for the determination of drug-albumin

affinity constants while simultaneously providing information on the location of the

drug binding site. Analyses of drugs in post-ingestion for select studies which provide

structural, spectral, and/or analytical data above and beyond routine toxicological

"screening" techniques. The forensic analysis of illicit drugs has been the subject of a

number of minor review articles and monographs over the past 10 years (1,2). In

addition, several articles have given more general overviews of the field (3).

Systematic approaches to substance identification have also been presented (4), and a

number of scientific working groups are currently establishing national/international

standards for forensic analysis of illicit drugs (5).

1

1.1. DRUG ANALYSIS

The assay analysis is essential and important in all the pharmaceutical

industries and quality control laboratories. Newer improved procedure for the

determination of pharmaceutical samples becomes inevitable.

1.2. ELECTROCHEMISTRY IN DRUG ANALYSIS

The historical developments of voltammetric and polarographic methods [6]

have revealed that these techniques are exceptionally useful in determining the main

component of a sample. In general, polarographic methods make themselves useful in

assay of pharmaceutical in tablets, injections, various solutions etc. It is convenient to

use voltammetric method to determine a substance, which is present even at a very low

concentration; its concentration being usually approximately known and interfering

substances are only seldom present. In contrast to optical methods such as

spectrophotometry, the voltammetric analysis can be done even in suspensions

containing insoluble particles of the excipient.

In 1970's, a radical change in the applicability of voltammetric methods in

drugs control took place by the introduction of pulse polarographic methods [7] by

which very low concentration about 10-6 mol l-1 could be measured. The usual micro

methods such as gas chromatography, high performance liquid chromatography with

different detectors as well as photo and fluorimetry are available for drug analysis in

vitro and in biological samples. But however almost all micro methods have certain

restriction in applications. For instance, in the case of gas chromatography the

limitation is determined by the volatility of the samples. Reactions can be used to

2

convert substances of high molecular weight to more volatile and stable derivatives,

which make them amenable to gas chromatography analysis.

HPLC, is clearly not a method for analytical problems with a high repetition

rate because the receptive conditioning of the system requires 24-36 hours. On the

other hand, electroanalysis is a manageable method, which is suitable for various

problems such as determination of intermediate in production control, detection of

impurities and by- product in pharmaceutical field. It would be an advantage if drugs

and or their metabolites in biological samples (blood, urine, bile and tissues) could be

analysed directly. However, this has only been achieved with radio immuno assay

procedures, and in some cases, with voltammetric methods, but with detection limits

(20 g ml-1)

The possibility of electrochemical determination of minocycline on mercury

and solid electrodes by various voltammetric and polarographic techniques has been

undertaken by Tanase et al. The possibility of using polarographic and voltammetric

techniques for minocycline analysis as simple and accurate methods have been

discussed [8]. Linear scan voltammetry, differential pulse voltammetry and

voltammetry with adsorptive accumulation at a glassy carbon paste electrode based on

mixing glassy carbon spherical microparticles with an organic pasting liquid have been

used for the determination of trace amounts of carcinogenic 1-nitropyrene and 1-

aminopyrene using a Britton-Robinson buffer – methanol mixture as a base electrolyte

[9]. The main advantage of this new type of electrode was its full compatibility with

media containing a high amount of organic solvent.

3

Ion transfer of the drug, d-tubocurarine at the interface of nitrobenzene / water

has been studied by polarography and cyclic voltammetry with due consideration to

the dissociation of d-tubocurarine [10]. Differential pulse voltammetry and Fourier

transformed infrared spectroscopic techniques have been applied and compared for

determination of azithromycin in two different pharmaceutical formulations (capsules

and suspension) by Araujo et al. [11]. Niyazi Yilmaz et al. have reported the

voltammetric determination of salbutamol based on electrochemical oxidation at

platinum and glassy carbon electrodes. The oxidative behavior of salbutamol has been

studied as a function of pH at platinum and activated glassy carbon electrodes and it is

used for the determination of salbutamol in pharmaceutical dosage forms [12]. The

surface of an electrochemical glassy carbon electrode was modified with a layer of

double-stranded DNA or with double-stranded DNA conditioned in single-stranded

DNA and was used to investigate mitoxantrone-DNA interactions by Voinea et al.

[13].

The voltammetric behaviour of meloxicam has been studied using direct

current, differential pulse polarography and cyclic voltammetry. The influence of

several variables has been examined in differential pulse polarographic method for

meloxicam. It is concluded that the developed method was accurate, sensitive, precise,

reproducible and useful for the quality control of meloxicam in pharmaceuticals and

spiked plasma [14]

Vieira et al. have reported the electrochemical and UV-Vis spectroscopic

properties of a series of pyridazine and 3,6-disubstituted pyridazine derivatives [15].

The interaction of mitoxantrone with calf thymus double-stranded DNA and calf

4

thymus single-stranded DNA was studied electrochemically by using differential pulse

voltammetry and cyclic voltammetry at a carbon paste electrode [16].

Hailemichael Alemu has reviewed the voltammetric behavior of drugs at the

interface between two immiscible electrolyte solutions. In his review, the results of the

electrochemical investigations made on the transfer of ionizable drugs at the interface

between two immiscible electrolyte solutions in the last decade have been presented.

Such studies point out the complexity of the distribution of ionizable compounds and

offer a new approach to relate the structure of such compounds to their passive

transport across biological membranes [17]. Al-Ghamdi et al. have developed a

sensitive and reliable stripping voltammetric method to determine cephalothin

antibiotic drug. This method was based on the adsorptive accumulation of the drug at a

hanging mercury drop electrode and then initiating a negative sweep which yield a

well defined cathodic peak at -625 mV using Ag/AgCl reference electrode. The

applicability of this approach is illustrated by the determination of cephalothin in

pharmaceutical preparation and biological fluids such as serum and urine [18].

1.2.1. Stripping Voltammetry for Pharmaceutical Analysis

It may be assumed that the application of stripping technique will grow,

especially in connection with the Pharmaceutical control. Further development of the

method depends on basic research, which tends to be concentrated in several main

areas. There are still many possibilities for the development of this relatively new

method of trace analysis. A few of them are:

Progress of the study of electrode reaction kinetics at the solid electrodes

5

Adsorption phenomenon of electrochemistry in non-aqueous media and

Development of chemical instrumentation which will enlarge the list of

methods of monitoring the stripping process and will allow extensive

automation of the method.

These voltammetric methods are applied in organic analysis most

frequently in pharmaceutical chemistry and pharmacology, in polymer chemistry, in

the foodstuff industry, in criminology and more recently in environmental research

[19].

1.3. ELECTROANALYTICAL TECHNIQUES

Most of the applications of environmental analysis involve trace determination

of the compounds, often at a ppb level or even lower. The techniques used in trace

determinations must lead to high sensitivity, sufficient selectivity, precision and

accuracy. These criteria are satisfied by electroanalytical techniques.

These methods are effective for environmental research because they enable

immediate measurement of changes in the concentration of the compounds. Another

advantage is that several compounds can be determined simultaneously. Continuous

monitoring is also possible and systematic error caused by transport and storage of the

sample could be avoided. The cost per sample analysis is also lesser compared to

chromatographic methods. The choice of the method depends on the nature of the

compound to be determined, as well as on the sensitivity and selectivity requirements.

6

The methods commonly used are voltammetry, polarography, potentiometry,

coulometry and conductometry. The sensitivity limits of common electroanalytical

methods are presented in the table 1.

Table 1. Sensitivity limits of electroanalytical methods

S. No. Techniques

Sensitivity

limits(M)

1 AC polarography, Thin layer coulometry 10-4 to 10-5

2 Chronocoulometry, Classical polarography 10-5 to 10-6

3

Derivative polarography, Square wave

polarography,Linear sweep voltammetry, Chemical

stripping analysis

10-6 to 10-7

4 Pulse polarography, Amperometry, Conductivity 10-7 to 10-8

5Anodic stripping with hanging mercury drop

electrodes.10-8 to10-9

6Anodic stripping with thin film electrodes or solid

electrodes.10-9 to 10-10

Hence electroanalytical techniques are applicable to a very large number of

organic compounds encountered in many fields. The literature survey for the last 25

years shows that polarography and voltammetry have been used in the organic field,

particularly in the pharmaceutical and biological fields [20]. Meites and Zuman have

7

listed the polarographic behaviour of a large number of substances [21]. Many

compounds that are neither reduced nor oxidized in the available potential range or for

which the signals acquired are not suitable for analytical purposes can be converted

into electroactive substances via chemical or electrochemical methods and then they

can be analysed [22,23].

1.4. SIGNIFICANCE OF VOLTAMMETRY

It has been established that voltammetry is a potent analytical tool in

environmental trace studies. Hence, a suitable voltammetric method has become one

of the preferred approaches in environmental trace analysis. Voltammetry leads to

extraordinary determination sensitivity with inherent high accuracy, i.e. small

tendency of systematic errors.

The following are the important advantageous features of voltammetry.

The simultaneous determination of several analytes by a single scan is often

possible with voltammetric procedure.

It has a reasonable high determination rate. Voltammetry equals or even

surpasses the analysis rate of atomic adsorption spectroscopy which is

considered as a more sensitive and accurate method [24,25].

The present introduction of automation into voltammetry will further enhance

convenience of application in routine analysis for the determination rate [26].

The instrument is very compact and is easily used in the field studies carried out

in ships or in mobile terrestrial areas.

8

Voltammetry is essentially a substance-specific and not just an element-specific

method like the other non-electrochemical methods.

1.4.1. Cyclic Voltammetry

Cyclic voltammetry is a technique that allows one to scan the potential of

working electrode either in anodic or cathodic direction and observe peaks due to

oxidation or reduction of the analyte. Then the potential scan is reversed in the

cathodic or anodic direction. The peaks due to oxidation and reduction of

intermediates formed during the forward scan may be observed. The electrode system

in cyclic voltammetry is dictated by the nature of the medium as well as the process

being studied. The commonly used electrodes are glassy carbon, planar platinum disks,

and platinum wires, hanging mercury drop, graphite and carbon paste. It is a simple

technique and provides a great deal of information about electrochemical behaviour.

Hence it is considered as one of the most powerful electrochemical diagnostic tools.

The potential may be swept anodically or cathodically and unlike polarographic

waves, the curves obtained are peaks [27]. One of the outstanding features of cyclic

voltammetry is its ability to generate a potential reactive species and then to examine it

immediately by reversal [28], thereby providing an electrochemical overview for a

reaction system.

The chief strengths of cyclic voltammetry are:

Applicability to a wide range of electrode materials.

A range of five orders of magnitude in scan rates.

Great flexibility in setting up scan limits and reversal conditions.

9

An intrinsic facility for highlighting the chemical conditions between various

electroactive species present in the voltammogram.

Highly developed theory.

The shape of the voltammogram depends strongly on the mechanism of the

electrode process. Cyclic voltammetry can provide information about the number of

electrons transferred in each peak. The diagnostic criteria for two important systems

are discussed and others are presented in table 2.The peak current for the reversible

process at 25 C is given by Randles-Sevcik equation [29].

ip = 2.69 x 105 n3/2 A D1/2C1/2

Where ip is the peak current in amperes, n is the number of electrons involved in

the reaction, D is the diffusion coefficient of the oxidant or reductant in cm2 sec-1, A is

the area of the electrode in cm2 and is the scan rate in Volt sec-1

The potential difference between Ep and Ep/2 is given by

Ep – Ep/2 = 56.5/n mVat 25C

The difference between Epa and Epc is given by

Epa – Epc = 59/n mV at 25C

For a reversible process, the anodic peak current ipa is equal to cathodic peak

current ipc and hence ipa / ipc is unity and is independent of [30].

Table 2. Diagnostic criteria for the charge transfer reactions

10

System Diagnostic criteria

Reversible

Ox + ne- Red

Ep is independent of ; Eo = (Epa – Ep

c) / 2

Epc- Ep

a = 59/n mV at 25o C and is independent of

ip/1/2 is independent of ; ipa/ ip

c is unity and independent of

Wave shape is independent of

Quasi reversible

(low v)Red-ne+Ox

Red-ne+Ox ( high v)

Ep shifts with v

Epc – Ep

a may approach 60/n mV at low but increases as increases

ip/1/2 is virtually only for = 0.5

Irreversible

Red-ne+Ox

No current response in reverse scan

Ep shifts cathodically by 30/n mV per tenfold increase in

The wave shape is determined by and is independent of

Preceding reversible chemical reaction

Z fk

bkRed

ne-

Ox

Ep shifts anodically with an increase in

ip/1/2 decreases as increases

Following reversible chemical reaction

Redne-

+Oxkb

kf

Ep shifts cathodically with an increase in

ip/1/2 virtually constant with

ipa/ ip

c decreases from unity as increases

Charge transfer with catalytic regeneration

Ox + ne-Red + Z

kc

Ep shifts anodically by a maximum of 60/n mV

ip/1/2 increases at low values of and becomes independent in higher

ipa/ ip

c is unity

Following irreversible dimerisation reaction

Z

Redne-+Ox

kRed2 d

Ep shifts cathodically by 20/n mV per tenfold increase in and per tenfold decrease in initial concentration, C*

ox

ip/1/2 decreases a maximum of 20% from low to high

ipa/ ip

c increases with and decreases as C*ox increases.

The peak current and the peak potential for an irreversible process are given by

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ip = 2.98 x 105 n [n]1/2 AD1/2 C

p = E0 – RT/nF [0.78 – 2.3/2 log (nFD/ KRT )] – 2.3 RT/2nF log

Hence, Ep shifts with scan rate according to

dEp / dlogn

In cyclic Voltammetric experiments, no anodic or cathodic peak would be

noticed in the subsequent cathodic or anodic sweep for an irreversible process. ip/1/2 is

independent of scan rate while the peak shifts cathodically as the scan rate increases

for an irreversible system.

Apart from cyclic voltammetry, other techniques used in electrochemical

studies are differential pulse voltammetry, square wave voltammetry,

chronocoulometry, controlled potential coulometry and stripping voltammetry.

1.4.2. Chronocoulometry

In this technique, the potential excitation function is stepped from an initial potential,

where no redox reaction occurs to a final potential where the reaction of interest does

occur, instead of measuring the current directly, it is integrated and the charge is

measured.

It offers several advantages [31]. They are as follows:

The later part of the response which is more accessible experimentally, is least

destroyed by non-ideal potential rise and offers better signal to noise ratios

while retaining the information from the early response,

Integration eliminates random noise and

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Contributions from diffusional and interfacial components are easily separated.

The forward chronocoulometric response of diffusing reactants is described by

the integrated Cottrell equation.

Qd = 2nFAD1/2Ct1/2 /

Where Qd = charge in coulombs, n = equivalent/mole, F = Faraday constant, 96485

c/equivalent, A = Area of the electrode, cm2 D = Diffusion coefficient, cm2/s, C =

concentration, mol/cm3 and t = time, seconds.

1.2.6. Controlled potential coulometry

Controlled potential bulk electrolysis or coulometry is often referred to as a

steady state technique which is used to determine the overall number of electrons

involved in the reaction. It is used to prepare reaction products which are then

identified by the application of conventional analytical techniques. A large electrode

area to solution volume area is desirable for this technique.

On the basis of steady state or sweep voltammetry, a certain reaction potential

under investigation will be at mass transport controlled rates. The current and its

integral, the charge is monitored as a function of time, usually until the current drops

to about 1.0%of its initial value. The most significant piece of information that is

obtained in the coulometric experiment is the value of n, the number of electrons

involved in the overall reaction.

13

Q = nFN

Q = total charge consumed, F = Faraday constant, 96485 c, n = number of

moles of electroactive species present and n = number of electrons involved.

1.2.7. Stripping Voltammetry

The electrochemical stripping analysis involves a preconcentration of the analyte on

the working electrode prior to its determination by means of an electrochemical

technique [32, 33]. It is a more important technique in trace analysis, since it has the

lowest detection limit in trace analysis. The original method involves the cathodic

electrodeposition of amalgam forming metals on a hanging mercury drop working

electrode, followed by the anodic voltammetric determination of the accumulated

metal during a positive signal potential scan [34]. In 1980s and 1990s several advances

have been made in the development of alternative schemes which further enhanced the

scope and power of stripping analysis [35, 36]. Consequently, numerous variants of

stripping analysis exist currently which differ in their method of accumulation and

measurements [37-40]. Stripping voltammetry enables the determination of

electroactive components in the concentration range from 10-6 to 10-9 M/dm3.

Research on increasing sensitivity of electroanalytical methods has led to the

development of the techniques of stripping voltammetry. The concentration step is

carried out for a definite time under reproducible conditions and the stripping process

in most cases is performed in some voltammetric scanning procedure. The resulting

“stripping voltammogram” shows peaks, the heights of which are generally

proportional to the concentration of the corresponding electroactive species and the

14

potentials of which have the same qualitative meaning as their half-wave potentials in

polarography. Dilute solutions in the range 10-6 to 10-9 M/dm3 and less, are analysed

with excellent precision and selectivity. Thus this technique extends the range of

classical polarography by three to four times making possibly the analysis in nano

range.

The important characteristics of stripping voltammetric peaks are its height,

width and peak potential. They are affected by the type of electrodes and scan rate.

The same electrode is used both in the concentration and stripping processes. The

process is not disturbed by the presence of organic substances other than analyte,

provided they are not adsorbed on the electrode surface.

1.2.7. 1. Differential pulse voltammetry

In differential pulse, the excitation wave form consists of small amplitude

pulses (10-100 mV) super imposed upon a staircase wave form. The major component

of the current difference is the faradaic current, which flows due to an oxidation or

reduction at the electrode. The capacitive current component due to the electrical

charging of the double layer is largely removed. Because of this, DPV gives higher

signal to noise ratios than other DC methods for quantitative analysis. The current is

sampled both just before application of the pulse and at the end of the pulse. The

output is the current difference plotted versus the base potential. The pulse amplitude

is constant with respect to the base potential. The base potential is not constant but is

scanned in small steps. The important parameters in this voltammogram are the peak

15

potential and the peak current. Many heavy metals and organics have been determined

by this pulse technique up to the range of 10-7 M to 10-9 M range.

1.2.7.2. Square wave voltammetry

Square wave voltammetry is a large amplitude differential technique in which a

waveform composed of symmetrical square waves superimposed on a base staircase

potential. The current is sampled twice during each square wave cycle, once at the end of the

forward pulse and once at the end of the reverse pulse. The reverse pulse causes the reverse

reaction of the product (of the forward pulse). Sensitivity increases from the fact that the net

current is larger than either the forward or reverse components (since it is the difference

between them). The total current response depends on both the reduction and the reoxidation

currents. The major advantages of square wave volammetry are its sensitivity, speed, fine

shape and position of the peak and easy repetitive monitoring. As a result, the analysis time is

drastically reduced and sensitivity is increased high. In order to compare the results found in

the CV studies, the voltammetric experiments were carried out with the square wave

technique also.

1.2.8. Stripping voltammetry for environmental control

It may be assumed that the application of stripping technique will grow, especially

in connection with the environmental pollution control. Further development of the

method depends on basic research, which tends to be concentrated in several main

areas. There are still many possibilities for the development of this relatively new

method of trace analysis. A few of them are:

Progress of the study of electrode reaction kinetics at the solid electrodes

16

Adsorption phenomenon of electrochemistry in non-aqueous media and

Development of chemical instrumentation which will enlarge the list of

methods of monitoring the stripping process and will allow extensive

automation of the method.

1.3. WORKING ELECTRODES

It is established that the rate of electrochemical process is extensively

influenced by the catalytic activity of the electrode surface; both the catalyst and the

supporting material are significant factors. Glassy carbon electrode and various

chemically modified electrodes are used as working electrodes in the present study.

1.3.1. Glassy Carbon Electrode

Nowadays, a variety of carbon materials have been employed as electrode in

electroanalytical studies [41]. Among these, glassy carbon is a specific variety of

synthetic carbon material, which was first synthesized, by Yamada and Sato [123] in

1962 by controlled heat treatment of premoulded polymeric phenol-formaldehyde

resin up to 3000C in an inert atmosphere. Structural studies showed that glassy carbon

is made up of perfectly smooth aromatic ribbons, which can stack above each other

forming microfibres. However, by choosing the proper moulds, this material may be

prepared in different forms like carbon fibres, disks, rods and rings. Its maximum pore

size is around 100nm [42]. From the earlier studies, it was realized that GCE materials

prepared at high heat-treatment shows good electrochemical activity [43,44]. Glassy

carbon thus prepared has exemplary features. It is stable to heat upto 3000C, it is

highly resistant to high temperature, air oxidation and chemical oxidizing agents. It

17

may even be boiled in nitric acid without noticeable loss of weight. Its gas

permeability is very low (10-9 cm2 s-1). GCE is polished using alumina in the form of

emery paper or powder, which activates it in electron transfer reactions. This

behaviour has been investigated thoroughly using electrochemical as well as other

microscopic techniques [45-49] Wang et al. [50] recommended a pretreatment

procedure that employs AC polarization. The most important pretreatment procedure

involves cycling between selected anodic and cathodic potential regions at a fairly

slow sweep rates, 10mV/s for 10-15 minutes [51] The aim of pretreatments is to

increase the number of active sites, that is, to increase the site density. Furthermore,

pretreatments introduce functional groups containing oxygen on carbon surfaces,

which may serve as mediators.

The advantageous features of GCE are:

Light weight and high mechanical strength

High resistivity to heat

High resistivity to chemicals

Absolute gas impermeability like glass

Excellent thermal and electrical conductivity

Less or no contamination on its fine impermeable structure

Chemically modified electrodes, which promise extensive applications in

electrosynthesis, electroanalysis and electrochemical energy conversion can also be

prepared on GCE [52-54].

1.9.2. Clay Modified Glassy Carbon Electrode

18

Clay minerals are cheap, widely available naturally occurring materials. Their

well defined layered structures [55-57] flexible adsorption properties [58] and

potential as catalysts and / or catalyst supports [59-63] make them interesting materials

with which to modify electrode surfaces. They have also higher thermal and chemical

stability than nafion and other polyelectrolytes [64].

Clay modified electrodes are prepared by the deposition of clay films on a

conductive substrate. The aim is to take advantage of the adsorption and / or catalytic

properties of these films to improve the selectivity or the sensitivity of the electrodes

toward solution species. Clays are heterogeneous materials and each individual clay

has a range of different compositions and particle sizes. Moreover, clay films are

imperfect stacks of clay layers. They contain many defects such as holes and pores of

various sizes. Hence they provide a number of sites for the adsorption of analyte [65].

Imperfections in the stacking of the clay layers result in holes, pores and other defects

in the films where the adsorbed species could be found. With more defects, the

probability that clay-bound cations have access to the electrode surface is increased.

Clay minerals used as modifiers belong to the class of phyllosilicates-layered

hydrous aluminosilicates. Their layered structure is either formed from on sheet of

SiO4 tetrahedra and one sheet of AlO6 octahedra (1:1 Phyllosilicates) or an Al-

octahedral sheet is sandwiched between two Si-tetrahedral sheets (2:1 Phyllosilicates).

A positive charge deficiency of layers is balanced by exchangeable cations (Na+, K+,

NH4+ etc.) bound on the external surfaces for 1:1 phyllosilicates and also in the

interlayer in the case of 2:1 phyllosilicates. A distance between the layers is an

important characteristic of clay mineral and it depends on the number of intercalated

19

water and exchangeable cations within the interlayer space. The important properties

of phyllosilicate structure such as large specific surface, ion-exchange properties and

ability to sorb and intercalate organic compounds (intercalation) predetermine

phyllosilicates, especially a group of smectites for preparation of clay electrodes

[83]67. Montmorillonite (MM) is the most often used smectite. Its cation exchange

capacity is typically 0.80-1.50 mmol g-1, anion exchange is about four times lower.

Thixotropy is a key physical feature that predetermines montmorillonite to be used as

stable and adhesive clay film. HPMM/GCE gives better response only in the presence

of surfactants. Sodium ion present in the clay matrix increases the conductivity.

Hence, NaMM/GCE is employed as a modified electrode in the present investigation.

The unit cell formula for montmorillonite in sodium from [66] is

[(Si7.84Al0.16)(Fe3+0.26Al3.22Mg0.4Fe2+

0.12)O20(OH)4Na0.68].

In general, the clay coated electrodes are more suitable for physico-

chemical studies-charge or ion transport the clay membrane, photocatalysis,

electrocatalysis etc.

1.9.3. Polypyrrole Modified Glassy Carbon Electrode

Conducting polymers exhibit good electrical conductivity. The modification of

electrodes with conducting organic polymers improves the electrodes’ sensitivity and

selectivity. The multilayered dynamic polymeric coating provides three dimensional

20

reaction zones at the electrode surface. It leads to increase the flux of reactions, which

in turn increases the sensitivity. Besides, the chemistry of interactions on such polymer

modified surfaces has been well pronounced in chromatography.

Among the conducting polymers so far produced, based on polyanilines,

polypyrroles, polythiophenes, polyphenylenes and poly (p-phenylene vinylene) have

attracted much attention. Recently, electrodes whose surface modified with conducting

polymers especially polypyrrole find extensive applications [67,68]

1.9.3. PEDOT modified Glassy Carbon ElectrodeConducting polymers have very good electronic conductance. Electrodes

modified with conducting organic polymers improved their sensitivity and selectivity.

Their ability to detect electro organic species can be realised using appropriately

modified surfaces [69,70]. Such improvements have been achieved by producing

modified surfaces that provide more efficient preconcentration, excluded interference

enhances the rate of electron transfer or produces unique, non-faradaic signals [65].

The multilayered dynamic polymeric coating provides three dimensional reaction

zones at the electrode surface. This leads to increase the flux of reactions, which in

turn increases the sensitivity. In addition to the above, the chemistry of interactions on

such polymer modified surfaces have been well pronounced in chromatography.

Of the many conducting polymers so far produced, based on poly anilines,

polypyrroles, polythiophenes, polyphenylenes and poly(p-phenylene vinylene) have

attracted much attention [71]. Recently electrodes’ whose surfaces modified with

21

conducting polymers especially PEDOT, a derivative of poly thiophene have recently

found extensive applications [72-78]. AG research laboratories in Germany developed

PEDOT, poly(3,4-ethylene dioxythiophene) [79].

PEDOT has the advantages of providing very stable and highly conducting

films [80-83]. It was found to be almost transparent in their oxidised films and

showed a very high stability in the oxidised state [84 85]. PEDOT possesses

remarkable electrochromic properties [86] and has recently been used in the

development of electro-luminescent diodes [87]. Its transparent films are used as

catalytic supporter for the proton exchange fuel cells [88]. Electrooxidation of PEDOT

was studied in non-aqueous and aqueous media [89]. Electrosynthesis of these films

and their applications as electrode materials are also reported [90-93].

1.9.4. Riboflavin Modified Glassy Carbon Electrode

Riboflavin belongs to the flavin family. Flavoenzymes are biologically

important compounds that take part in the electron transport chain of the respiratory

cycle, which involves oxygen reduction. The redox behaviour of flavin molecules [94]

in electro analysis has led to the modification of various electrode surfaces [95-101]

Since they form thermodynamically reversible systems, they are used as

electron transfer mediators. They undergo electron exchange readily with the analyte

and again at electrode surface and thereby acting as a shuttle to transport electrons

22

between the analyte and the electrode surface. The modifications of flavin have been

done by adsorption [102-104] and covalent linkage [105]

Riboflavin is neutral in both the oxidized and reduced states. Hence, the

addition or diffusion of any counter ion is not necessary in the redox conversion of this

compound in contrast to other redox mediators. Ep of reversible redox peaks of

riboflavin is 65 mV only. It undergoes two electron redox reactions on the electrode

surface [106]. The electrodes modified with riboflavin act as good catalysts in the

electro analysis of fine chemicals and dyes.

1.9.5. Polyaniline Modified Glassy Carbon Electrode (PANI/GCE)

Conducting polymers exhibit good electrical conductivity. The modification of

electrodes with conducting organic polymers improves the electrode sensitivity and

selectivity. The multilayered dynamic polymeric coating provides three dimensional

reaction zones at the electrode surface. It leads to increase the flux of reactions, which

in turn increases the sensitivity.Among the conducting polymers so far produced,

based on polyanilines, polypyrroles, polythiophenes, polyphenylenes and poly (p-

Phenylene vinylene) have attracted much attention.

CHAPTER II

STATE OF THE ART AND SCOPE OF THE WORK

STRUCTURE OF CEFEPIME

23

Chemical data

Formula C19H24N6O5S2 

Mol. mass 480.56 g/mol

Pharmacokinetic data

Bioavailability 100% (IM)

Metabolism Hepatic 15%

Half-life 2 hours

Excretion Renal 70–99%

2.1 TRADE NAMES

Neopime(Neomed)

Maxipime,

Maxcef,

Cepimax,

Cepimex

Axepim

2.2. IMPORTANCE

24

Cefepime is usually reserved to treat severe nosocomial pneumonia, infections

caused by multi-resistant microorganisms (e.g.Pseudomonas aeruginosa)

and empirical treatment of febrile neutropenia.  Cefepime has good activity against

important pathogens including Pseudomonas aeruginosa, Staphylococcus aureus, and

multiple drug resistant Streptococcus pneumoniae. A particular strength is its activity

against Enterobacteriaceae. Whereas other cephalosporins are degraded by

many plasmid- and chromosome-mediated beta-lactamases, cefepime is stable and is a

front line agent when infection with Enterobacteriaceae is known or suspected. The

combination of the syn-configuration of the methoxy imino  moiety and

the aminothiazolyl moiety confers extra stability to β-lactamase enzymes produced by

many bacteria. The N-methyl pyrrolidine  moiety increases penetration into Gram-

negative bacteria. These factors increases the activity of cefepime against otherwise

resistant organisms including Pseudomonas aeruginosa and Staphylococcus aureus.

2.3 SIDE EFFECTS

Less serious side effects may include:

pain, swelling, or skin rash where the injection was given;

stomach pain, nausea, vomiting;

headache;

skin rash or itching;

white patches or sores inside your mouth or on your lips; or

vaginal itching or discharge.

25

Serious side effects

confusion, hallucinations, or convulsiones o se desmaya. seizure (black-out or

convulsions);

diarrhea that is watery or bloody;

skin rash, bruising, severe tingling, numbness, pain, muscle weakness;

fever, chills, body aches, flu symptoms;

easy bruising or bleeding, unusual weakness; or

sore throat and headache with a severe blistering, peeling, and red skin rash.

2.4 STUDIES OF CEFEPIME

Cefepime, a fourth generation cephalosporin, is a broad spectrum antibiotic

with improved activity against gram-negative bacteria over other commercially

available cephalosporin drugs. It is a valuable option in the treatment of lower

respiratory tract infection, urinary tract infections, skin and skin structure infections,

bacterial meningitis. Analysis of cefepime purity is challenging due to isomeric and

other impurities with similar structures. Additionally, cefepime is particularly labile

and its stability is highly pH dependent, in part due to rapid N-methylpyrrolidine

(NMP) cleavage at room temperature.

Isla et al., Developed a new rapid and reproducible HPLC method for the

determination of cefepime and ceftazidime in plasma and dialysate-ultrafiltrate

samples obtained from intensive care unit (ICU) patients undergoing continuous veno-

venous hemodiafiltration (CVVHDF)[107]. Ocana Gonzalez et al. [108] developed a

optimised method for the simultaneous determination of the cephalosporin cefepime

26

and the quinolones garenoxacin, levofloxacin and moxifloxacin. Identification and

quantification was carried out with a diode-array UV detector, with working

wavelengths of 256 nm for cefepime The retention times and detection limits for

cefepime was found t be 4.9min and 1.9µg/mL−1 respectively. Siddiqui et al. [109]

developed a simple and sensitive assay method for simultaneous determination of

cefepime and sulbactam sodium in Supime. With UV detection at 230 nm.

Chromatographic separation of two drugs was achieved on a Hypersil ODS C-18

column using a binary mixture of acetonitrile and tetrabutyl ammonium hydroxide as a

mobile pha se adjusted to pH 5.0. Vicente Ródenas et al. [110] described a simple

spectrophotometric assay for the determination of cefepime and L-arginine in

injections. Liu and his co-worker Bruce [111] found a and accurate capillary zone

electrophoresis (CZE) method for simultaneous determination of cefepime and L-

arginine in cefepime for injection. Best results were achieved with the background

electrolyte (BGE) prepared by titrating 40 mM sodium dihydrogen phosphate with

phosphoric acid to pH 2.3 and an applied voltage of 30 kV in a bare fused-silica

capillary. The capillary temperature was 30°C and detection was made at 195 nm.Five

drugs of pharmaceutical interest cefepime hydrochloride, cefoperazone sodium,

ceftazidime pentahydrate, cefuroxime sodium and etamsylate were determined

Spectrophotometrically using ammonium molybdate by Marwa et al [112]. Ocaña

González et al simultaneously determined cefepime and grepafloxacin in human urine

by HPLC.Detection wavelengths were 259 nm for cefepime and 278 nm for

grepafloxacin. The retention times were 4.03 min for cefepime and 8.85 min for

grepafloxacin, with detection limits of 1.0 and 1.1 microg mL(-1), respectively [113].

Valassis et al.[114] have determined quantitatively cefepime in plasma and vitreous

27

fluid by HPLC. Palacios et al. [115] studied adsorptive stripping voltammetric

determination of cefepime in human urine and cerebrospinal fluid on the hanging

mercury drop electrode, followed by linear sweep voltammetry (staircase). They have

reported that the drug was strongly adsorbed in acid media, with maximum adsorption

at pH 5.8. The detection limit found was 4.8×10−10 M, with 120-s preconcentration.

Breilh et al. determined cefepime and cefpirome in human serum by HPLC using an

ultrafiltration for antibiotics serum extraction [116]. Christine Farthing and his co-

workers determined Cefepime and Cefazolin in Human Plasma and Dialysate by

HPLC.They utilized a C18 column with an aqueous mobile phase of dibasic potassium

hydrogen phosphate (pH 7.0) and methanol gradient at a flow rate of 1 mL min−1. The

method demonstrated linearity from 2.0 to 100.0 μg mL−1 (r > 0.999) with detection

limit of 1 μg mL−1 for both cefepime and cefazolin[117]. During preparation and

storage, cefepime degrades by release of the N-methylpyrrolidine (NMP) side chain

and opening of the beta-lactam ring. Brian De Borba and  Jeff Rohrer of Dionex

Corp., USA [118]have determined N-Methylpyrrolidine in Cefepime Using Reagent-

Free Ion Chromatography. Theydescribed an improved method using a hydrophilic,

carboxylate-functionalized cation exchanger with suppressed conductivity detection to

determine NMP in cefepime. Compared to the current USPmethod, the proposed

method significantly reduces the time between injections, thereby improving sample

throughput. Souza and co-wrkers [119] developed a microbiological assay for

determination of cefepime in injectable preparations. The paper reports the

development and in-house validation of an agar diffusion bioassay using a cylinder-

28

plate method for the determination of cefepime in powder for injection. The validation

performed yielded good results in terms of linearity, precision, accuracy, and

robustness. Saeed et al.[120] developed an accurate, sensitive and least time

consuming RP-HPLC method for the estimation of cefpirome in the presence of

essential and trace metal.UV detection was performed at 265 nm. The detection limit

of cefpirome was 10 ng. Drug metal interaction studies were carried out at 37 oC to

monitor the complexation of drug with metal ions like Mg(II), Ca(II), Cr(II), Mn(II),

Fe (III),Co(II), Ni(II), Cu(II), Zn(II) & Cd(II). The order of complexation was ferric >

chromium > copper > nickel > cadmium > zinc > magnesium > manganese >calcium >

cobalt. The antimicrobial activity of cefepime and rifampicin in cerebrospinal fluid

was ssudied by Richard et al. [121]. They have reported that bacterial killing was

achieved by cefepime at lower drug concentrations. S.Majdi et al. studied the

electrochemical oxidation and determination of ceftriaxone on a glassy carbon and

carbon-nanotube-modified glassy carbon electrodes.They have reported that

Ceftriaxone can be determined with a detection limit of 4.03×10−6 M [122].

SCOPE OF THE WORK

Cefepime remains an important benchmark for new antibiotic drug

development, there is increasing need for sensitive and high throughput determination

of cefepime in biological matrices to support preclinical, clinical pharmacokinetic, and

toxicokinetic studies

29

Electrochemical studies of cefepime assume importance because of the

presence of electroactive carbonyl, thio, ketone, acid and amine group. Among various

antibiotic drugs, cefepime is important because of its utility in the field of

pharmaceuticals. Hence this compound is chosen for the present study.

One of the most useful features of cyclic voltammetry is its ability to generate a

potentially reactive species and then to examine it immediately by reversal. Cyclic

voltammetric technique provides the electrochemical overview for a reaction system.

Among the various electrochemical methods, stripping voltammetry is very

much useful in sample identification because of their high sensitivity, which is in the

parts per million (ppm) range. The organic compounds such as pesticides, herbicides

and drugs can be determined in parts per billion (ppb) level. Hence, these

electrochemical techniques are used in this study.

Of many electrodes used in electrochemical studies, gold is known to give quite

good reproducible responses next to mercury and glassy carbon electrode. Gold is a

well-behaved electrode material for the study of many reactions over a wide range of

potentials. Hence these electrodes are employed in this study.

The objectives of the present investigation on cefepime are:

To study the electrochemical behaviour

To study the effect of pH on the oxidation

To optimize of differential pulse stripping voltammetric parameters

30

To propose electroanalytical procedure for the determination of

antibiotic.

The experimental details of the present study are outlined in chapter III. The

results and discussions are presented in chapter IV. The conclusion of the work is

summarized in chapter V

31

CHAPTER -III

EXPERIMENTAL DETAILS

3.1. INSTRUMENTATION

The electrochemical analyzer from CHI 660C was employed for various

electrochemical studies performed. This instrument uses the latest analog and

microcomputer design to provide high performance, better precision and greater

versatility in electrochemical measurements. This instrument was employed for

performing voltammetric studies, and differential pulse stripping voltammetry

32

3.2. CELL SETUP

The cell was made of glass, having a capacity of 15ml and the teflon made cell

top was comprised of three separate holes for the insertion of electrodes viz., working

electrode, counter electrode and reference electrode. The cell top also has the purging

and blanketing facilities for nitrogen gas with separate tubes to remove oxygen gas.

This setup enables to maintain an inert atmosphere above the sample solution

throughout the experiment. These functions are controlled through CHI 760C

software.

3. 3. WORKING ELECTRODE

3. 3. 1. Gold Electrode and pretreatment

The gold electrode used in the present study whose geometric area of cross

section is 0.0314cm2. Freshly polished and cleaned gold electrode surface contains

surface functions that show reversible redox behaviour. In order to get reproducible

results great care was exercised in the electrode pretreatment.

33

First the electrode was washed with water- ethanol- ammonia mixture followed

by ethanol- acetic acid and ethylacetate- ethanol mixture. It was then washed with

distilled water and trichloroethylene. With this method, oxides and surface-active

substance and organic compounds were removed from the electrode surface when

electrode surface was seriously contaminated. The most effective and at the same time

the simplest way followed for the renewal of the surface was to remove a layer of it by

rubbing with fine powder of alumina. After rubbing, the electrode is wiped with filter

paper and then rinsed with water. The fine particles of alumina adsorbed on electrode

surface were removed by ultrasonication in presence of water.

The electrode showed a well- defined wave for the ferro/ferri cyanide system.

Hence, the reproducibility of results was verified by recording cyclic voltammograms

of ferricyanide systems and especially measuring the peak separation.

Ep = Epc - Epa.

From the earlier studies it has been established that after polishing and cleaning the

electrode was potentiodynamically swept between -0.5 to + 1.25 V in the medium for

30 minutes at a slow sweep rate of 40 mV s-1 gave reproducible cyclic

voltammograms of ferricyanide system with a EP value quite close to 60 mV. Similar

activation procedure was found to give reproducible behaviour in the present study as

well and hence this method was employed to activate the electrode throughout the

present work.

3. 3. 2. Reference and counter electrodes

34

Ag/Ag+ electrode was used as reference electrode in this investigation. A

platinum wire was used as counter electrode. Pt wire electrode was cleaned

successively with dilute detergent solution, isopropyl alcohol and sodium hydroxide

solution. Finally it was rinsed with distilled water. All electrochemical experiments

were performed at 25 + 0.1oC.

3.4. REAGENTS AND CHEMICALS USED

cefepime was obtained from Sigma and used. 0.1M solution was prepared by

dissolving the compound in water. Supporting electrolytes of different pH were

prepared.

1. 0.1 M H2SO4 (pH 1.0)

2. B.R Buffer (pH 4.0)

3. 0.1 M KCl (pH 7.0)

4. B.R Buffer (pH9.2)

5. 0.1 M NaOH (pH 13.0)

ELCO 1120 pH meter was used to check the pH values of the buffer solutions.

3.5. EXPERIMENTAL PROCEDURES

3. 5. 1. Cyclic Voltammetry

Before each set of experiments, the electrochemical cell was scrupulously

washed with nitric acid, double distilled water and dried in an air oven. The glassy

carbon electrode was preconditioned. The counter electrode platinum was cleaned

35

with nitric acid. Reference electrode SCE was also thoroughly washed with double

distilled water. The solution of 10ml was placed in the electrochemical cell. The three

electrodes were inserted into the cell and purified nitrogen gas was purged for 20

minutes to remove the dissolved oxygen under stirred conditions. All electrochemical

studies were carried out by thermosetting the electrochemical system at 25 + 1oC

For each pH condition of a system in a medium, preliminary experiments were

carried out to assess the potential range where the electrochemical process was taking

place. Since the electrode surface at the starting potential was employed in each

experiment, background current was measured at various sweep rates. For recording

the cyclic voltammograms of the substance, suitable aliquots of the stock solution

were mixed with appropriate electrolyte solution to get 10ml final volume of the

required concentration. The solution was stirred and nitrogen gas was purged for 20

minutes. Then cyclic voltammograms were recorded at various sweep rates and at

various concentrations of the substrate. The reproducibility of the voltammogram was

often verified by recording the responses under identical conditions with various

intervals of time. Whenever the reproducibility was lost, the electrode was removed

and complete pretreatment procedures like polishing, washing and potential cycling

were repeated to get the reproducible results.

In analytical determination data, care was taken to subtract the background

current in each sweep rate.

3. 5. 2. Chronocoulometry

36

Chronocoulometric experiments were carried out in a similar type of cell used

for voltammetric studies. The chronocoulometric behaviour of the analyte chosen was

studied at all selected pH.

The test solution was purged with nitrogen. The potential was stepped up from

an initial potential to a final potential. A pulse width of the range 2 to 10 seconds was

maintained. The initial potential was chosen, where no redox reaction occurred and the

final potential was chosen where the reaction of interest was over. The cyclic

voltammetric data obtained were used as the criterion to fix these potentials. Instead of

measuring the current directly, it was integrated and the charge was measured. The

plot of Q vs. t1/2 was found to be a straight line and the slope of this line is written as

2nFAD1/2C / 1/2 which was derived from Cottrell equation. By substituting the known

parameters in the above equation, unknown parameter n, the number of electrons

transferred was arrived by known D value or vice versa.

3. 5. 4. Controlled Potential coulometry

Controlled potential coulometry is readily performed with EG & G

electrochemical analyzer. The electrochemical cell as well as the electrode system was

the same as utilized in cyclic voltammetry studies. Micro molar to nano molar

solutions of the compound was chosen as the concentration of the substrate. In

aqueous medium the study was performed at different pH condition as studied in CV

experiments.

37

From cyclic voltammetric data, the potential of electrolysis was chosen for the

compound. Initially the electrolytic solution containing no substrate was taken under

nitrogen atmosphere and the charge was observed. This charge was then subtracted

from the total charge consumed, as the former was a base charge due solely to buffer

components. After the addition of the substrate, it was completely electrolysed by

applying a fixed potential throughout the experiment.

When the experiment was on, the software exhibited the values of current and

charges consumed and indicated the duration of the experiment also. For each second,

the current and charge were updated on the screen. From the output of Q vs. time

curve, the total charge consumed for the electrolysis was measured and was used to

calculate the number of electrons transferred per molecule by means of Faraday’s law.

Q = n F N

Where Q = charge in Coulomb; n = number of electrons transferred and N = number

of moles / ml of the substrate

3. 5. 2. Differential Pulse Stripping Voltammetry

Measurements were made using differential pulse voltammetry for confirmation

of CV studies by following the similar procedure to that of CV studies. Differential

pulse stripping voltammetric studies were employed for the analytical study. Dilute

solutions were preferred for stripping process. In differential pulse, the excitation wave

form consists of small amplitude pulses (10-100 mV) super imposed upon a staircase

wave form. The major component of the current difference is the faradaic current,

38

which flows due to an oxidation or reduction at the electrode. The capacitive current

component due to the electrical charging of the double layer is largely removed.

Because of this, DPV gives higher signal to noise ratios than other DC methods for

quantitative analysis. The current is sampled both just before application of the pulse

and at the end of the pulse. The output is the current difference plotted versus the base

potential. The pulse amplitude is constant with respect to the base potential. The base

potential is not constant but is scanned in small steps. The important parameters in this

voltammogram are the peak potential and the peak current. Many heavy metals and

organics have been determined by this pulse technique up to the range of 10-7 M to10-9

M range.

39

CHAPTER IV

RESULTS AND DISCUSSION

ELECTROCHEMICAL STUDIES OF CEFEPIME

Electrochemical studies were carried out for cefepime at gold electrode and the

results were discussed in detail.

4. 1. ELECTROCHEMICAL BEHAVIOUR OF CEFEPIME ON GOLD

ELECTRODE

Cyclic voltammetric behaviour of cefepime in pH 1.0, 4.0, 7.0, 9.2 and 13.0 on Gold electrode was presented in Figure 1-5. The cyclic voltammogram exhibited one well-defined oxidation peak. The background current was recorded for all pHs, studied in the potential range 0 to 1.6 (vs.Ag+) and subtracted properly in calculating the peak

currents. The values of peak current and peak potential are presented in Table 3.

Fig.1 Cyclic voltammetric behaviour of cefepime in pH 1 medium

40

Fig.2 Cyclic voltammetric behaviour of cefepime in pH4 medium

Fig.3 Cyclic voltammetric behaviour of cefepime in pH7 medium

41

Fig.4 Cyclic voltammetric behaviour of cefepime in pH9.2 medium

Fig.5 Cyclic voltammetric behaviour of cefepime in pH13 medium

42

Table 2. Cyclic Voltammetric behaviour of cefepime at different pHs

pHE(V)

ipx10-4(A)

1.0 1.084 1.042

4.0 1.145 1.700

7.0 1.131 1.353

9.2 1.110 1.300

13.0 1.118 1.510

4.2. EFFECT OF pH

Cyclic voltammetric studies of cefepime at five selected pH revealed electro

active nature and one oxidation peak in all pHs. The peak potential is around 1.0 V it

may be oxidation of primary amine group in cefepime. The peak potentials and

currents were measured for the oxidation peaks and correlated with pH to understand

the influence of pH on the oxidation behaviour of cefepime. Figure 6 shows the

variation of peak current with pH and this resulted in curve. Maximum peak current

for oxidation was observed at pH 4.0. This may be due to faster electron transfer rate

at lesser acid medium pH 4 and this indicates that the rate of the reaction is controlled

only by electron transfer. Figure 7 shows the non-linear variation of peak potential

with pH and it has not resulted in a straight line. Lowest peak potential for oxidation

is observed at pH 1.0 but higher current was observed at pH4.0. This may be due to

easier oxidation i.e. the electrochemical energy required for the oxidation processes is

43

minimum at pH 4.0. From the study it is concluded that pH 4.0 is the best pH for

further cyclic voltammetric studies and the development of electro analytical method

for the determination of cefepime.

Fig.6 Plot of peak current Vs pH

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10 12 14

pH

ipx1

0-4

A

Fig.7 Plot of peak potential Vs pH

1.08

1.09

1.1

1.11

1.12

1.13

1.14

1.15

0 2 4 6 8 10 12 14

pH

E(V

)

44

4.3. DETAILED ELECTROCHEMICAL STUDIES OF cefepime ON GOLD

ELECTRODE AT pH 4.0

Cyclic voltammogram obtained at pH 4.0 (Fig 7) also exhibited one anodic

peak. The results obtained are given in table 4. The effect of scan rate was studied by

varying the scan rate from 50 to 200 mV/s at a concentration of 0.002 M in pH 4.0.

The peak current increased gradually for oxidation peak.

The peak potential of the anodic peak shifted little anodically.

The correlations were made for the well-shaped anodic oxidation peak observed

in the forward scan. The peak current increased linearly with sweep rate (Fig.9) and

less correlation. A non-linear plot ip Vs. 1/2 at 0.002M concentration is presented in

figure 10. The slope of the straight line (y=0.5975x-0.7515) obtained from the plot log

ip vs. log (Fig.11) was 0.5975. These facts revealed that the voltammetric redox

process was adsorption-controlled.

45

Fig.8 Electrochemical studies of cefepime on gold electrode in pH 4.0 medium at

varying scan rates

Table 3. Cyclic voltammogram data of cefepime on gold electrode

scan rate

Conc E( V) ip(A)

50

0.0020

1.145 1.9

75 1.148 2.3

100 1.151 2.7

125 1.154 3.1

150 1.157 3.5

175 1.160 3.9

200 1.163 4.2

50 0.0034 1.022 2.1

0.0046 1.066 2.3

0.0058 1.088 2.7

46

0.0060 1.121 2.9

Fig.9 Peak Current Vs scan rate

y = 0.0156x + 1.1393

R2 = 0.9987

0

1.5

3

4.5

0 50 100 150 200 250

ipx

10

-4 A

Fig.10 peak current Vs square root of scan rate

y = 0.332x - 0.5439

R2 = 0.9938

1.000

2.500

4.000

5.500

6.0 10.0 14.0

ipx1

0-4

A

47

Fig.11 Log peak current Vs log scan rate

y = 0.5975x - 0.7515

R2 = 0.9918

0.20

0.50

0.80

1.5 1.8 2.1 2.4

log

log

ip

4.4. IRREVERSIBILITY

In the reverse scan, no cathodic peak was observed. This shows the

irreversibility of electron transfer. The irreversibility was further confirmed by

calculating n values from the slope of the straight line plot EP Vs log (Fig. 12). The

slope value was substituted in the following expression for the irreversible charge

transfer

-dEp / dlog = -30/n

n is found to be 0.1031. The fractional value of n confirms irreversible

electron transfer.

48

Fig.12 Plot of peak potential Vs log scan rate

y = 30.952x - 33.662

R2 = 0.9389

1.0

2.0

3.0

1.140 1.150 1.160 1.170

log

E(V

)

4.5. EFFECT OF CONCENTRATION

As the concentration of the compound increased from 0.002M to 0.01M at a

constant sweep rate of 50 mV/s, there was a gradual increase in the peak current with

little variation in the peak potentials (Fig 13). This again confirmed the adsorption-

controlled reaction. All the above facts revealed that electro oxidation of cefepime

using gold electrode at pH 4.0 was adsorption-controlled and irreversible reaction.

49

Fig.13 Plot of peak current Vs Concentration

y = 1563.5x + 14.393

R2 = 0.9921

0

5

10

15

20

25

30

35

0 0.002 0.004 0.006 0.008 0.01 0.012

Conc(M)

ipx1

0-5

(A)

4.3. Chronocoulometry

3.17x10-8 M/cm3 concentration of cefepime in 50% aqueous ethanol medium

was taken for chronocoulometric experiment. The potential range was chosen in

between 883 and 1242 mV. The plot of Q vs. t1/2 obtained and used to calculate.

Diffusion coefficient, D was calculated from the value of forward slope obtained and

was found to be 5.531x10-5 cm2/s for pH 4.0. The same experiment was repeated for all

selected pH values.

4. 4. Controlled potential coulometry

Controlled potential coulometry was carried out in the five selected pH media.

The number of electron transferred was calculated by employing Faraday’s law. The

exhaustive electrolysis was carried out for cefepime of concentration 2.72x10-9 M/dm3

at pH 13.0. The potential was maintained at 1240 mV. The charge vs. time plot was 50

used to determination of number of electron. From charge obtained, ‘n’ the

coulometric number of electrons transferred was calculated. It was found to 2e-

oxidation.

4. 5. Reduction Mechanism

The experimental evidences obtained proved that the electrochemical oxidation

was dependent on pH of the medium. In all pH media, the oxidation preceded by 2e -

reactions. Based on the above discussion and the mechanism proposed for cefepime

the following probable oxidation mechanism is proposed.

N

SH

OCOOH

N

N

OS N

NH2

N

OCH3

-2e-

N

SH

OCOOH

N

N

OS N

NHOH

N

OCH3

4. 6. ELECTROANALYTICAL DETERMINATION OF CEFEPIME

4.6.1 DIFFERENTIAL PULSE STRIPPING VOLTAMMETRY

Adsorptive stripping voltammetry involves two steps in which the first step is

accumulation of the substrate on the electrode and the second step involves stripping.

Cyclic voltammetric results revealed the adsorption of the substrate on gold electrode

at pH 4.0. It was expected that cefepime would be adsorbed on the electrode during the

accumulation step and stripped off easily in the stripping step. Hence, cefepime was

considered as a suitable substrate for the adsorptive stripping voltammetric studies.

Cefepime exhibits a very good stripping signal. A systematic study of various

51

instrumental parameters that affect the stripping response has been carried out with 10 -

2 M of drug to establish the optimum conditions.

4.6.1.1. Effect of deposited potential (Eacc)

Hence optimization of the accumulation potential was done as the first part for

this study. This potential was varied from 0.2 to 1.0V at an accumulation time of 15s.

Maximum Peak current was observed at 0.2 V and it was fixed as the optimum

accumulation potential.

4.6.1.2. Effect of deposited time (Tacc)

By varying the deposition time from 15 to 35 seconds, the effect of deposition

time was studied after fixing the deposition potential at 0.2 V. The results are

presented in table 4. This table reveals highest current is observed at 15s deposition

time. Hence this is taken for the further consideration.

4.6.1.3. Effect of initial potential (Ein)

The initial scanning potential is another important parameter as it confirms the

non-faradaic nature of the preconcentration step. It also controls both the peak

potential and peak current in the stripping voltammogram. The influence of the initial

potential on the peak current was studied by varying the initial scan potential from 0 to

0.8 V. The peak current is affected by this initial potential in a different way. Better

response ie, high peak current with better resolution was observed at 0.2 V. Hence 0.2

52

V was fixed as initial scanning potential and the reproducibility of the method was

determined by making successive measurements.

4.6.1.4. Effect of pulse Amplitude (PA)

Effect of pulse amplitude was studied by varying from 25 to 150mV. The sharp

peak current was observed at the pulse amplitude of 100mV.

4.6.1.5. Effect of pulse width (PW)

Pulse width also studied by varying from 25 to 150mV. The sharp peak nature

with higher current was observed at pulse width of 100mV. Hence the pulse width of

100mV was chosen as optimum value.

4.6.1.6. Effect of scan increment (SI)

The scan increment was varied between 4 to 16mV and maximum peak current

response was obtained at 4mV scan increment.

4.6.1.7. Effect of pulse period (PP)

After fixing the above parameters, effect of pulse period was studied. PP was

varied from 2 to 10 sec. highly resolved stripping response was obtained at the PP of

2sec.

4.6.1.8. Optimum condition:

All the experimental parameters are optimized as discussed above (Table 5).

The range of study and the optimum values are arrived at table 6.

53

4. 6. 2. CONCENTRATION DEPENDENCE

The dependence of the peak current on concentration was studied under the

above fixed optimum parameters. Experimental results showed that peak current

increased with increasing concentration of cefepime. A representative differential

stripping voltammogram obtained for cefepime was given in the figure 14. The

derived calibration plot (Fig 15) indicated the linear dependence of peak current with

concentration. The limits of concentration were 2.43902x10-09 to 1.11111x10-06 M. The

reproducibility of the stripping signal was realized in terms of relative standard

deviation for ten identical measurements carried out at a concentration level of 1x10 -5

M and the RSD was found to be 2.7%. The lower limit of detection was 2.439x10-11 M.

Table 5. Differential pulse stripping voltammetric behaviour cefepime on Gold electrode at pH 4.0

Parameter Eacc DT Eis PA PW SI PP

Cefepime

Ep (V)ip (10-

5A)

Accumulation potential (Eacc) V

0.2

15 0 25 50 4 4

0.977 6.86

0.4 1.019 6.63

0.6 1.023 6.30

0.8 0.968 6.64

1.0 0.991 6.52

Deposit time (DT) sec

0.2 15 0 25 50 4 4 0.988 7.77

20 1.013 6.19

25 1.075 6.45

54

30 1.125 6.62

35 1.320 6.86

Initial scan potential (Eis) V

0.2 15

0

25 50 4 4

1.042 6.23

0.2 0.977 6.66

0.4 1.025 6.55

0.6 0.956 6.15

0.8 1.023 6.55

Pulse amplitude (PA) mV

0.2 15 0.2

25

50 4 4

1.043 3.01

50 1.011 9.70

75 0.938 15.00

100 0.920 16.13

150 0.968 14.21

Pulse width (PW) mSec

0.2 15 0.2 100

25

4 4

0.918 20.42

50 0.922 23.04

75 0.936 29.75

100 0.945 64.75

150 0.947 51.33

Scan Increment (SI) mV

0.2 1.5 0.2 100 100

4

4

0.977 89.25

8 0.975 88.33

10 0.981 84.17

12 0.986 77.51

16 0.975 76.04

Pulse period (PP)

Sec

0.2 1.5 0.2 100 100 4 2 0.952 91.88

4 0.949 83.81

6 0.950 78.37

55

8 0.942 75.00

10 0.931 74.63

Table 6. Optimum experimental conditions of cefepime by differential pulse

stripping voltammetry

Variables Range examined

Optimum value

Deposition potential, V 0.2-1.0 0.2

Deposition time, S 15-35 15

Initial scanning potential, V 0-0.8 0.2

Pulse amplitude, mV 25-150 100

Pulse width, mS 25-150 100

Scan increment, mV 4-16 4

Pulse period (PP) S 2-10 2

Fig.14 DPSV of cefepime under optimum experimental condition

56

Fig.15 Plot of peak current Vs conc.

y = 1E+07x + 16.53

R2 = 0.9979

15

20

25

30

35

0.00E+00 2.00E-07 4.00E-07 6.00E-07 8.00E-07 1.00E-06 1.20E-06Conc (M)

ip(

A)

CHAPTER – V

CONCLUSION57

Electrochemical behaviour of cefepime was studied in detail and an electro

analytical procedure was developed. The cyclic voltammetric studies at different pH

media and different concentration to ascertain the redox behaviour of cefepime in an

undivided cell and differential pulse stripping voltammetric studies to propose an

analytical method to determine cefepime have been carried out.

Gold electrode surfaces have been chosen as working electrodes for the present

study.

This investigation clearly establishes the reproducibility of the usage of gold

electrode under all experimental conditions.

Care has been exercised throughout the studies to keep the electrode surface

fresh always. Hence for every sweep rate changes in cyclic voltammetry, a clear

polished gold electrode has been employed.

Conclusions from Cyclic voltammetric studies

1) In general one anodic peak was observed in all pH media studied. The oxidation

peak did not satisfy the criteria for reversibility with the cathodic peak in the

reverse scan. Hence, the oxidation and reduction were irreversible electron

transfer has been proposed.

2) The study on the influence of pH on the redox behaviour of cefepime revealed

maximum peak current at pH 4.0. This could be explained due to faster electron

58

transfer rate and easier oxidation of cefepime at this pH. The results from this

study at gold electrode suggested the selection of pH 4.0 for the electrochemical

studies.

3) Anodic peak showed straight line with good correlation in almost all cases, when

ip values are correlated against ½ at all concentrations and ip values against C at

50 mVs-1 scan rate. The peak current function showed constancy and led to a

conclusion that electron transfers are controlled only by adsorption.

4) Chronocoulometry and controlled potential coulometry were carried out to

determine the number of electrons involved in the oxidation. It resulted as two

electrons in all pH media studied.

5) On the basis of the cyclic voltammetric and coulometric results, a probable

reaction mechanism is proposed.

From Stripping voltammetric studies:

1. Differential pulse stripping voltammetric (DPSV) studies were carried out at

optimum pH 4.0 for cefepime using the gold as a working electrode.

2. Optimum experimental conditions were arrived by varying the parameters such as

accumulation potential, accumulation time, initial potential, pulse height, pulse

width, and scan increment.

59

3. The peak current was measured under optimum conditions at various

concentrations. The concentrations were directly proportional to the stripping

peak current. The ip and concentration were correlated and the result was a

straight line. The calibration plot was arrived at and used for the determination of

cefepime. The analytical characteristics are found out and presented.

4. The lower limit of detection of cefepime using the proposed stripping

voltammetric method was 25 ppb. The relative standard deviation obtained for 7

identical measurements was 2.7% and it reveals good reproducibility of the

stripping method.

Thus an electro analytical determination procedure was developed and proposed

in this investigation for cefepime. The application of this method for the

determination of cefepime in tablets, pharmaceutical formulations, urine and blood

samples and checking the validity of the proposed method are the scope of the future

work.

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